An update on the distribution of Blastocystis subtypes in the Americas

Blastocystis is an intestinal protist that presents worldwide distribution, colonizes animal and human hosts, and is classified into at least 34 ribosomal subtypes (STs). Herein, we conducted an update based on studies reporting Blastocystis-positive samples obtained from diverse hosts in the Americas. We described the distribution throughout the continent by assembling maps representing the distribution of STs and the most important 18S-rRNA alleles. Thirty-nine articles from the previous study, “A summary of Blastocystis subtypes in North and South America,” and forty-one additional articles from March 2019 to March 2022 were considered. The most common subtype described was ST3, representing the highest percentage of positive samples. Other recently identified STs include ST12, ST13, and ST16 in humans, and ST10, ST14, and ST17 in animals. Novel subtypes have also been described in this continent. We assembled and updated the distribution of Blastocystis in the Americas. We hope this delivers new understandings and knowledge of this microorganism’s prevalence and genetic diversity.


Introduction
Blastocystis is a stramenopile known as the most common enteric protist in humans and animals worldwide [1,2]. Likewise, its extensive distribution in developed and developing countries has been described [3,4]. It colonizes multiple host digestive systems, and the parasite transmits through the fecal-oral route, through water, foods, and some animals are considered the most probable means of spread [4,5,6,7,8]. Due to its high frequency in different species of animals, the hypothesis of zoonotic transmission of Blastocystis between animals and humans has recently been recognized [2,5,9,10,11,12,13,14,15]. Although, its zoonotic potential and transmission should be considered in further studies [16].
Blastocystis is known to cause gastrointestinal infection in humans, but it is not very well known whether Blastocystis infection causes gastrointestinal symptoms in animals. However, Blastocystis infection has been associated with gastrointestinal illnesses, urticaria, and other extraintestinal signs of the inflammatory response [9,17,18,19,20,21]. It has also been reported in asymptomatic patients [22,23,24]. Indeed, the relationship between disease and colonization remains unclear. The high prevalence of polyparasitism in the Americas accompanied by nonspecific symptoms does not allow associations between clinical manifestations and the presence of the microorganism or even link it to any ribosomal subtype.
This eukaryotic microorganism has been recognized as an essential component of the gut microbiota. It is suggested that Blastocystis role could be related to gut homeostasis by maintaining a higher gut bacterial diversity [3,20,25,26,27,28]. Other reports revealed that Blastocystis could be a healthy member of the gut microbiota, and its interaction with other microbial communities might regulate the host immune responses [22,29,30,31,32,33,34]. Regardless of studies reporting a decrease in the abundance of beneficial bacterial communities in the gastrointestinal tract or intestinal dysbiosis due to the presence of Blastocystis [22,23,30,34,35].
Currently, there are known 28 Blastocystis Subtypes (STs) whose assignment is based on sequence analysis of the small subunit of ribosomal RNA (rRNA-18S) gene and phylogeny using a numbering system based on publication date [16]. Various tools have been used for its subtyping; these tools include partial amplification of the 18S-rRNA gene, a barcoding method that uses specific primers from a 600 bp region of length [36,37,38], and next-generation amplification sequencing by Illumina targeting a variable region of the equal length [39,40]. Also, recent approaches have focused on third-generation sequencing using the MinION by Oxford Nanopore Technologies, the first tool that uses nanopore machinery to obtain complete sequences (1800 pb) from the Blastocystis 18S-rRNA gene [16,41,42,43].
In recent years, Blastocystis subtyping has evolved to obtain complete sequences of the 18S-rRNA gene, allowing a robust classification to categorize new subtypes, generate reference sequences, and build accurate phylogenetic lineages [16]. Furthermore, these molecular methods have demonstrated a considerable genetic diversity among Blastocystis inter and intra-subtype levels; some studies reported that comparing sequences in different hosts could show similar or highly different strains in some STs. Within the described STs, some have been linked to specific hosts. Of the 28 STs reported worldwide, twelve subtypes (ST1-ST10, ST12, ST13, ST14, ST16, and ST23) have been detected in humans and animals [44,45], ST1-ST4 the most frequent in human samples [46,47]. Mixed infections have been described in humans and animals, and multiple subtypes have been described within one sample [46,48,49,50].
The distribution of Blastocystis in the Americas is not yet fully clarified. However, in the last five years, there has been an increased interest in detecting and identifying Blastocystis STs in these countries, significantly impacting this protozoan's epidemiological and molecular characterization. Environmental aspects in the American continent, such as poverty, sanitation problems, poor access to potable water, internal civil conflicts, and high biodiversity, lead to a high prevalence of this intestinal microorganism which cannot yet be linked to intestinal manifestations, pathogenic potential, or beneficial influence on gut microbiota. Despite the progress, the epidemiological data obtained have not yet been consolidated. Therefore, this study aims to update the distribution of Blastocystis throughout the Americas from samples identified in humans and animals. Maps and graphs were constructed to identify the most frequent subtypes and alleles, including an ample discussion of the subtyping methods used.

Identifying available data
Considering our previous review, "A summary of Blastocystis subtypes in North and South America" [46], and conducting a new literature search by using databases such as PubMed, Science Direct, Scopus, and the Integrated Search System of Universidad del Rosario, Colombia, led us to find forty-one (41) additional articles from March 2019 to March 2022. In total, eighty (80) papers met the requirements to be considered for the development of this study. The keywords included Blastocystis, subtypes, STs, molecular characterization, epidemiology, distribution, alleles, genetic diversity, America, intestinal protozoa, and isolates.
This research was geographically limited to the American continent, including three subcontinents: North America, Central America, South America, and thirty-five nations. There were excluded those reports whose samples were taken outside the continent. We incorporated studies from different languages: English, Spanish, and Portuguese. Information was extracted from the articles containing the date of publication, country, and geographic location of sampling. Based on the information from those reports, we established the following inclusion criteria: methods of identification either by microscopy or molecular methods, subtyping including the use of Next-Generation sequencing, and the host from which the samples were obtained.

Data arrangement
Upholding the data extraction methodology of the previous paper, the information and data from each study were collected, including country, samples' exact location, methods of identification and subtyping, number of positive samples, host, subtype, identified alleles, authors (last name of the first author) and year of publication. The data extraction was performed in January and March of 2021 and 2022. The database of the previous study was used to update it with the latest information and complement the prior publication. The current information obtained from those studies which met the inclusion criteria was added to the variables. The samples' geographic location was also paired with their corresponding coordinates (latitude and longitude) from the collected places.
Using the arranged data, maps and graphics were constructed using R programming. Updating the distribution of Blastocystis STs throughout the American continent, including specific geographic hotspots of ST's occurrence in thirteen different countries, revealing the emergence of new subtypes and new locations of identification of this parasite. First, we built a map that contains all subtypes identified in the Americas, in both human and animal hosts. Subsequently, maps were constructed for the most frequent subtypes (ST1, ST2, ST3) and individually containing percentages of each subtype by country. This information was used to build graphs representing the number of alleles and frequency of Blastocystis subtypes in the Americas.

Data analysis
Chi-square tests were applied to identify possible associations between variables of interest (STs, hosts, and countries). Multiple comparisons between different categories were made by implementing post hoc tests through the chisq.posthoc.test function included in the vcd package R software implements pairwise comparisons using Bonferroni as an adjustment method. All statistical analyses were performed using the R software (RStudio Team 2019). All tests of significance were twotailed, and P-values < 0.05 were considered statistically significant.
Although ST1 is widely distributed in human samples throughout the continent, positive samples from animal hosts were limited to the following countries: Brazil [56,57,58,69], Colombia [43,77], M exico [97], and the United States [48,111]. The most common animal host were mammals such as pigs, dogs, cats, and white-tailed deer. Interestingly, ST2-positive samples from animal hosts were found only in primates from Brazil [58,69] and Perú [101]. On the other hand, ST3 positive samples distribution from an animal host was also limited across Brazil [57,58,69], Colombia [43], Perú [101], and the United States [10,48,111], being its significant representatives' of mammals and primates.
Other subtypes have limited distribution. Some subtypes have been described in only one country. ST12 has been described in human host samples only in Bolivia [47]; data analysis has shown a statistically significant association between Bolivia and this subtype (p ¼ 1.40E-05). Likewise, ST13 has only been described in human hosts from Peru [47]. ST16 in Colombian human hosts [31], and recently in this country, a new subtype (ST32) was identified in cows and goats [43]. Some unique subtypes, ST27, ST28, and ST29, were identified in wild bird species [39,71]. Furthermore, the latest studies on white-tailed deer in the United States demonstrated the presence of new subtypes, such as ST30 and ST31 [48]. Even though ST7 and ST8 had been reported in different countries, we found that they are correlated with Brazil (p ¼ 0.002955; p ¼ 6.00E-06).
It is important to recognize that samples in all the countries mentioned are reported in human hosts but not in all animal host samples described. Some countries such as Brazil [39,56,57,58,64,65,69,71], Colombia [43,77], Ecuador [87], Mexico [94], Peru [101,104], and United States [10,48,105,109,110,111], have demonstrated the presence of this protozoan in animals. Hence, the number of samples between humans and animals varies.

Blastocystis Alleles by ST
Considering the information obtained, sixty-two different alleles have been reported in Blastocystis subtypes in the Americas. It was found that the most frequent allele in animal and human hosts was ST3 (a34), present in 503 positive samples, followed by a36, reported in 397 positive samples from ST3, and a4 described in 300 positive samples from ST1. The alleles reported in each subtype are shown in Table S2 and below in Figure 4. Despite its wide distribution throughout the Americas, allele distribution is limited to countries such as Argentina, Brazil, Colombia, and the United States [9,24,31,47,56,59,62,64,67,68,69,70,76,77,83,101,112] (Figure 4; Table S2).
The alleles information was obtained from those studies, which included in their methodology allelic typing (n ¼ 16)-summarizing the information from those studies, which performed allele identification from both human and animal Blastocystis positive samples, in Table S2. Not every allele had a representative amount, and some alleles had less than 10% representation in each subtype. There are more samples without allelic identification in most STs, mainly in recent studies.

Discussion
Blastocystis is the most frequent enteric protozoan found in animal and human hosts worldwide [12,118]. Our findings suggest that the number of countries reporting Blastocystis-positive samples and its subtypes has increased in recent years, providing valuable information about new hosts, transmission patterns, and ecological aspects and helping to clarify genetic and evolutionary changes among subtypes.
Brazil, Colombia, Mexico, and the United States have been highlighted as the major representatives in terms of Blastocystis reports in the Americas [ Colombia is considered a heterogeneous country regarding geography, climate, ecosystems, and biodiversity. Its biogeographical regions have their features, including variations in socioeconomic conditions, health care, waste collection, sewage treatment, and cultural and behavioral factors. These considerations could be linked to the high frequency of intestinal pathogens due to factors that may favor their transmission [83,121]. According to data from the Institute of Hydrology, Meteorology and Environmental Studies (IDEAM -Instituto de Hidrología, Meteorología y Estudios Ambientales), population growth has driven an expansion of economic activities, which influences the reduction in the availability of resources in the country [122]-leading to a disturbance in the ecological niches where some pathogens may be found, possibly promoting their propagation, and affecting their circulation.
Brazil, Mexico, Argentina, and Ecuador [24,33,40,47   also contributed a large number of positive samples for ST3, ST1 and ST2 exceeding one hundred samples per country. Interestingly, we observed a correlation between human hosts and the subtypes mentioned. This suggests a particular role of these STs in the Americas, and their preponderant distribution might be related to particular regional features. More studies are needed to identify the biological properties and their implications for virulence and disease outcomes.
Previous studies in Europe, Asia, and Oceania highlighted the genetic diversity of this protozoan. Numerous reports in European countries have shown that ST4 is the most common subtype in human samples [36,44,124,125]. However, it is also reported a high prevalence of ST1, ST2, and ST3 in Europe, Australia, and Southeastern Asian countries [44,126,127]. There can be contrasting realities within the countries that conform to each continent. Nemati et al. published an overview of Blastocystis subtypes reported in Asia and Australia, indicating that ST1 and ST3 were the most prevalent subtypes from human samples in Asian countries [128]; in southern countries such as Thailand, these subtypes were found in human samples as well [44].
Additionally, other subtypes have been described in this country (ST2, ST10, ST11, ST13, ST14) [128]. Unlike European countries, the actual source of ST4 is unclear in the southern Asian region; it is more common in East Asian countries and zoonotic subtypes ST6 and ST7 [44,129]. Based on these findings, the authors suggested that the transmission and distribution patterns may be affected by climate and geographic features, not only by socioeconomic conditions. As the tropical climate increases, the prevalence of this protozoan changes, which is reflected in the high frequency of some STs in eastern than western Asian countries [129]. The communities living close to their animals, belonging to a rural environment with a single water source, could explain the presence of previously undescribed subtypes in humans (ST23 and ST10), suggesting the importance of identifying the different routes of parasite transmission [44].
These differences between continents highlight the importance of further investigating the molecular distribution of Blastocystis to explain if ST3 is associated with transmission patterns between humans and identify the spreading mechanism of ST1-ST4 in non-human hosts. Also, it is essential to add new phenomena related to the distribution of Blastocystis and its subtypes, understanding these factors as climate, socioeconomic conditions, hydrographic distribution, water management, and treatment, among others that may be associated with the prevalence in different geographical locations.
Even though subtypes 1 to 9 are primarily described in humans, the information obtained from this review revealed that in smaller percentages, ST1-ST8 were found in animal hosts, including different species [10,43,48,56,57,58,69,77,94,97,101,105,109,111]. Some STs showed a strong correlation between specific animal hosts, particularly ST5-ST8 (Table S3). Our analysis showed several associations between the same subtype with different hosts, as well as it was demonstrated that the same host could have significant associations with multiple subtypes, one of them being cattle, which showed correlations with more than one Table 2. Animal Host species in Blastocystis positive samples.
Additionally, new subtypes have been described in the Americas, and most of them have been identified in animal hosts. Novel subtypes such as ST24, ST25, and ST27-ST29 were not previously reported in some countries such as Colombia and Brazil [39,71]. New subtypes in the United States, such as ST30 and ST31, were first detected in white-tailed deer, including ST21 and ST23-ST26 [48]. Higuera et al. reported subtypes ST21, ST23-ST26 in Colombian sheep, goats, horses, llamas, and cattle. Furthermore, they detected a previously reported new subtype, ST32, in goats and cattle [43]. These recent novel subtypes demonstrate an innovative approach to estimating Blastocystis genetic diversity and emerging subtypes. This suggests that using NGS (Next Generation Sequencing) strategies will facilitate our understanding of the molecular epidemiology of Blastocystis and the implications of mixed STs within individuals.
The distribution of Blastocystis in the Americas has exposed an increase in the number of reports in the last few years and a growing interest in identifying subtypes. This is supported by new methodological subtyping techniques such as next-generation sequencing and criteria to establish the nomenclature of novel subtypes [39]. Complete sequencing of the 18S-rRNA gene has opened the door to many possibilities for identifying subtypes. It could bring a closer look to elucidate the geographical and molecular distribution of the protozoan in a more precise and complete way, hence the importance of having more countries and researchers using these methods.
This work shows that the use of NGS has decreased the reports of "Novel ST" terminology in the last two years, increasing the identification of numerical classification of subtypes, including the description of new subtypes in North and South America [39,43,48]. Since the information on these novel subtypes is still limited to certain countries, the application of these methods has been reported mainly in Brazil, Colombia, and the United States, and the reports before 2019 use different molecular techniques. It should be considered to continue sequencing the complete gene (18S) to provide genuinely novel STs and determine the genetic differences between the detected STs throughout the continent. Lastly, as NGS continues to be employed by researchers across the Americas (sequencing capacity increased due to the worldwide efforts deployed for genomic surveillance during the COVID-19 pandemic), novel targets should be developed, or even Whole Genome Sequencing typing strategies should be in place for the better comprehension of Blastocystis genetic diversity.
Thirty-five nations integrate the American continent, and Blastocystispositive samples have been reported in thirteen countries. Despite the interest in the molecular identification of Blastocystis, more investigation is needed to explain the unknown data, to have a complete molecular distribution description of this protozoan, and encourage those twentytwo countries that have not yet reported or subtyped it. The previous statement could indicate that most countries of the continent are developing countries; access to new technology, molecular tools, and reagents can be expensive. Therefore, it can be considered a limitation in achieving molecular diagnosis and robust analysis in parasite research. Understandably, most of the reports in this study are performed by microscopy identification; those with access to advanced tools such as Next Generation platforms are limited.

Conclusions
In the last two decades, attempts have been made to demonstrate Blastocystis genetic variability and the differences between subtypes. Likewise, its prevalence in the world is also highly variable, drastically impacting its geographic distribution worldwide. This molecular and geographic variety has been shown in the American continent. Countries such as Colombia, Brazil, Mexico, Peru, and the United States have promoted the publication of new studies that support the update of valuable epidemiological data. However, the information obtained allowed us to update the existing subtypes but is insufficient to determine the current subtypes circulating in the Americas as a whole; this is only a slight approximation.
Additionally, we remark to consider in further studies environmental factors such as biodiversity, climatology, hydrographic, and sociodemographic, among others, which could explain the emergence of novel subtypes or changes in its distribution. We encourage countries that have already demonstrated the protozoan presence to conduct subtyping studies. Equally, we suggest applying new subtyping methods, such as the complete sequencing of the 18S-rRNA gene, to adequately detect new and currently circulating subtypes, maintaining the criteria to avoid typing errors or wrong molecular distribution. Each researcher in the Blastocystis community is responsible for maintaining an adequate Blastocystis nomenclature and promoting valuable information for the population, generating awareness of the existence and prompt identification of potentially pathogenic microorganisms such as Blastocystis. Finally, we call upon scientists to continue innovating methodologies and maintain an interest in identifying this parasite, whose importance lies in its high prevalence.

Declarations
Author contribution statement Paula Jim enez: conceived and designed the study; performed the study; analyzed and interpreted the data; wrote the paper.
Marina Muñoz: conceived and designed the study; analyzed and interpreted the data; wrote the paper. Juan David Ramírez: conceived and designed the study; performed the study; analyzed and interpreted the data; wrote the paper.

Funding statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement
Data included in article/supp. material/referenced in article.

Declaration of interest's statement
The authors declare no conflict of interest.

Additional information
Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2022.e12592.